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Regulation of TNF sensitivity via receptor modulation controlled by protein kinase c
2s
S-3
ESTROGEN RECEPTORS, DNA CONTENT ABNOR~~ALITIES AND CELL PROLIFERATION KINETICS IN MAMMARY CARCINOMAS Christov K., Tsingilev D., Todorov V., National Institute of Oncology, 1156-Sofia, Bulgaria Hundred eighty mammary carcinomas were investigated for estimation of any relationship between ER status, DNA aneuploidy (determined by flow cytometry) and cell proliferation kinetics (IH-, +14C-TdR autoradiography). The data obtained might be summarized as follows: 67 9,of mammary carcinomas are composed of DNA aneuploid cell lines. Seventy % of diploid tumors are ER+ against 67 9, in the non-diploid group. There is no relationship between DNA index (01) values and ER content. S-phase fraction and 3H-TdR incorporation are higher in ER - tumors than in ER + variants, Among the group of DNA diploid carcinomas ER negative variants indicate higher proliferative activity than ER positive tumors. Data are also presented for TS and TDP values.
S-5
REGULATIONOF TNF SENSITIVITY
VIA RECEPTOR MODULATION CONTROLLEO BY PROTEIN KINASE C. Thoma, B., Unglaub, R., Scheurich, P., and Pfizenmaier, K., Klinische Arbeitsgruppe der Max-Planck-Gesellschaft, 3400 Gottingen, FR Germany. TNF-~diated biological responses, e. g. cytotoxic effects or enhancement of HLA-antigen expression, are clearly dose-dependent and thus proportional to the number of ligand-receptor interactions. We were interested to investigate what mechanisms regulate TNF-receptor expression Binding studies with iodinated recombinant TNF-alpha and might thus control TNF-responsiveness. on various human cell lines revealed that activators of protein kinase C (pk-C) rapidly modulate TNF binding-capacity, rendering the cells TNF unresponsive. Specificity was confirmed using inhibitors of pk-C and by failure of phorbol esters to modulate TNF binding-capacity of isolated cell membranes. Occupation of TNF receptors with the ligand antagonizes with pk-C-induced receptor modulation. Further, pk-C activators do not stimulate internalization of TNF/receptor complexes. These findings and the very rapid time course of receptor modulation are consistent with a drastic affinity change probably due to a direct protein kinase C-mediated phosphorilation of TNF-receptors rather than internalization or shedding. Decrease in TNF-sensitivi-ty via down-regulation of TNF binding-capacity migh-t be important for poQentia1 TNF producer cells (e. g. monocyteslmacrophages), which are per se TNF sensitive, but can be stimulated by protein kinase C activators to produce TNF-alpha.
S-6 MECHANISM
OF GLUCOCORTICOID-INDUCED
Delfs T., Hoffmann Dept. Clin. Chem.,
Medical
LYMPHOMA
CELL DEATH
B., Freese V. and Wielckens Clinics,
University
K.
of Hamburg,
F.R.G.
Glucocorticoid action on certain lymphoma cells is characterized by a proliferation block followed by cell lysis. Corticosteroid-induced cell death cannot be demonstrated before 18-24h after glucocorticoid addition in S49.1 and 48-72h in L1210 lymphoma cultures, both of murine origin. The lymphoma cell lysis appears to involve the activation of an endonuclease leading to internucleosomal DNA strand breaks. In order to restore chromatin integrity DNA repair mechanisms are activated including the poly(ADP-ribosyl)ation system, a NAD dependent chromatin modification reaction involved in the maintenance of the chromatin structure during DNA repair. Consequently glucocorticoids decrease the NAD level in lymphoma cells. Combination of dexamethasone with the poly(ADP-ribosyl)ation inhibitor benzamide did not only increase the number of dead cells but also shifted the appearance of lysed cells to earlier times in both lymphoma cell lines pointing to a start of DNA fragmentation before cells actually loose their viability. The chromatin damage is continously reversed by repair processes until NAD is consumed. Then the DNA repair capacity decreases, within time leading to cell death. The decreased repair capacity is also reflected by altered repair kinetics following a challenge with an alkylating agent. Therefore it appears that the cytolytic effect of dexamethasone is mediated by at least two different mechanisms, an increase in DNA fragmentation by endonuclease activation and a decrease in the repair capacity.
S-3
ESTROGEN RECEPTORS, DNA CONTENT ABNOR~~ALITIES AND CELL PROLIFERATION KINETICS IN MAMMARY CARCINOMAS Christov K., Tsingilev D., Todorov V., National Institute of Oncology, 1156-Sofia, Bulgaria Hundred eighty mammary carcinomas were investigated for estimation of any relationship between ER status, DNA aneuploidy (determined by flow cytometry) and cell proliferation kinetics (IH-, +14C-TdR autoradiography). The data obtained might be summarized as follows: 67 9,of mammary carcinomas are composed of DNA aneuploid cell lines. Seventy % of diploid tumors are ER+ against 67 9, in the non-diploid group. There is no relationship between DNA index (01) values and ER content. S-phase fraction and 3H-TdR incorporation are higher in ER - tumors than in ER + variants, Among the group of DNA diploid carcinomas ER negative variants indicate higher proliferative activity than ER positive tumors. Data are also presented for TS and TDP values.
S-5
REGULATIONOF TNF SENSITIVITY
VIA RECEPTOR MODULATION CONTROLLEO BY PROTEIN KINASE C. Thoma, B., Unglaub, R., Scheurich, P., and Pfizenmaier, K., Klinische Arbeitsgruppe der Max-Planck-Gesellschaft, 3400 Gottingen, FR Germany. TNF-~diated biological responses, e. g. cytotoxic effects or enhancement of HLA-antigen expression, are clearly dose-dependent and thus proportional to the number of ligand-receptor interactions. We were interested to investigate what mechanisms regulate TNF-receptor expression Binding studies with iodinated recombinant TNF-alpha and might thus control TNF-responsiveness. on various human cell lines revealed that activators of protein kinase C (pk-C) rapidly modulate TNF binding-capacity, rendering the cells TNF unresponsive. Specificity was confirmed using inhibitors of pk-C and by failure of phorbol esters to modulate TNF binding-capacity of isolated cell membranes. Occupation of TNF receptors with the ligand antagonizes with pk-C-induced receptor modulation. Further, pk-C activators do not stimulate internalization of TNF/receptor complexes. These findings and the very rapid time course of receptor modulation are consistent with a drastic affinity change probably due to a direct protein kinase C-mediated phosphorilation of TNF-receptors rather than internalization or shedding. Decrease in TNF-sensitivi-ty via down-regulation of TNF binding-capacity migh-t be important for poQentia1 TNF producer cells (e. g. monocyteslmacrophages), which are per se TNF sensitive, but can be stimulated by protein kinase C activators to produce TNF-alpha.
S-6 MECHANISM
OF GLUCOCORTICOID-INDUCED
Delfs T., Hoffmann Dept. Clin. Chem.,
Medical
LYMPHOMA
CELL DEATH
B., Freese V. and Wielckens Clinics,
University
K.
of Hamburg,
F.R.G.
Glucocorticoid action on certain lymphoma cells is characterized by a proliferation block followed by cell lysis. Corticosteroid-induced cell death cannot be demonstrated before 18-24h after glucocorticoid addition in S49.1 and 48-72h in L1210 lymphoma cultures, both of murine origin. The lymphoma cell lysis appears to involve the activation of an endonuclease leading to internucleosomal DNA strand breaks. In order to restore chromatin integrity DNA repair mechanisms are activated including the poly(ADP-ribosyl)ation system, a NAD dependent chromatin modification reaction involved in the maintenance of the chromatin structure during DNA repair. Consequently glucocorticoids decrease the NAD level in lymphoma cells. Combination of dexamethasone with the poly(ADP-ribosyl)ation inhibitor benzamide did not only increase the number of dead cells but also shifted the appearance of lysed cells to earlier times in both lymphoma cell lines pointing to a start of DNA fragmentation before cells actually loose their viability. The chromatin damage is continously reversed by repair processes until NAD is consumed. Then the DNA repair capacity decreases, within time leading to cell death. The decreased repair capacity is also reflected by altered repair kinetics following a challenge with an alkylating agent. Therefore it appears that the cytolytic effect of dexamethasone is mediated by at least two different mechanisms, an increase in DNA fragmentation by endonuclease activation and a decrease in the repair capacity.